Identification of the immunophilin protein, FKBP12, as a specific receptor for the powerful immunosuppressant drug, FK506, led to a burst of pharmaceutical research over the past decade. Much of that research was aimed at the discovery of other high affinity ligands for FKBP which might become clinically and commercially significant immunosuppressant agents. A variety of synthetic FKBP ligands were produced and evaluated, but with disappointing results. In the early 1990's it was learned that FK506 works, not simply by binding to FKBP, but by mediating the association of FKBP with the effector protein, calcineurin, to form the tripartite complex which actually mediates immunosuppression. This finding explained the absence of immunosuppressive activity for the many high-affinity FKBP ligands which are now known in the art. A body of synthetic knowledge pertaining to the design and synthesis of FKBP ligands had been developed which appeared destined to lie fallow. See e.g., U.S. Pat. Nos. 5,192,773; 5,330,993; WO 92/19593; and WO 94/07858.
Largely independent of the search for new immunosuppressive agents, however, pioneering work on the design, production and use of biological switches based on ligand-mediated multimerization of recombinant proteins, including immunophilin-based fusion proteins, was reported. See Spencer et al, 1993, Science 262:1019-1024 and International Patent Applications PCT/US94/01660 and PCT/US94/08008. Spencer et al reported a new class of biologically active substances based on dimers of FK506, covalently attached to each other via a synthetic linker moiety. The resultant dimers (“FK1012” molecules) are characterized by high binding affinities for immunophilin molecules and are capable of mediating the association or complexation of fusion proteins containing FKBP domains. However, FK1012 and related semisynthetic multimerizing agents are large, complex molecules which can be inconvenient to produce.
New methods and materials for multimerizing chimeric proteins containing immunophilin moieties would be desirable, where the methods and materials involve smaller, simpler multimerizing agents which are more convenient to produce and which are more readily amenable to structural modification.
Important initial research based on N-oxalyl-pipecolyl and N-oxalyl-prolyl ligand moieties and aimed at providing wholly synthetic replacements for FK1012-type semi-synthetic multimerizing agents was disclosed in PCT/US95/10559. Further progress in this direction, including new multimerizing agents e.g. which are more conveniently prepared, have alternative pharmacokinetic profiles and/or which bind preferentially or with higher affinity to genetically engineered immunophilin domains relative to their binding to native immunophilin proteins would be very desirable.